1. Field of the Invention
The present invention relates generally to methods of protein expression and purification, and more specifically, to methods of expression and purification of immunotoxins.
2. Description of the Related Art
The number of organ transplants performed in the United States each year is approximately 24,000 and consists predominantly of kidney transplants (14,000), liver transplants (5,000), heart transplants (2,200), and smaller numbers of pancreas, lung, heart-lung, and intestinal transplants (2002 OPTN/SRTR Annual Report).
Transplant tolerance remains an elusive goal for patients and physicians whose ideal would be to see a successful, allogenic organ transplant performed without the need for indefinite, non-specific maintenance immunosuppressive drugs and their attendant side effects. Many of these patients have been treated with cyclosporin, azathioprine, and prednisone with a variety of other immunosuppressive agents being used for induction or maintenance immunosuppression. The average annual cost of maintenance immunosuppressive therapy in the United States is approximately $11,000 (Immunosuppressive Drugs Coverage Act, National Kidney Foundation, available at www.kidney.org/general/pubpol/ir_mufact.cfm). While these agents are effective in preventing rejection, the side effects of immunosuppressive therapy are considerable. Immunosuppressive therapy induces nonspecific unresponsiveness of the immune system. Recipients are susceptible to infection and there is a risk of malignancy such as in the form of post transplant lymphoproliferative disorders. A major goal in transplant immunobiology is the development of specific immunologic tolerance to organ transplants with the potential of freeing patients from the side effects of continuous pharmacologic immunosuppression and its attendant complications and costs.
A bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G4S) was developed for tolerance induction for transplantation, T-cell leukemia and autoimmune diseases. The immunotoxin consists of the first 390 amino acid residues of diphtheria toxin (DT390) and two tandem antigen-binding domains (sFv) from the anti-CD3 antibody UCHT1, that are responsible for binding the immunotoxin to the CDR3εγ subunit of the T cell receptor complex. The anti-CD3ε antibody moiety enables the immunotoxin to target specific cells and the diphtheria toxin moiety kills the target cells. The immunotoxin may be utilized to effect at least partial T-cell depletion in order to treat or prevent T-cell mediated diseases or conditions of the immune system.
Administration of an anti-T cell immunotoxin provides an approach for specific immunologic tolerance. It is applicable to new organ transplants and potentially to existing transplants in recipients with stable transplant function. The immunotoxin can provide highly specific immunosuppression and imparts transplant tolerance in primates, without the adverse effects of nonspecific immunosuppressive drugs, anti-lymphocyte serum or radiation. It is a goal in this field to inhibit the rejection response to the point that rejection is not a factor in reducing average life span among transplant recipients.
The methylotrophic yeast Pichia pastoris has been used successfully to express heterologous proteins from different origins (Gellissen 2000). As an eukaryote, Pichia pastoris has the ability to perform many post-translational protein modifications such as proteolytic processing, folding, disulfide bond formation and glycosylation. Like other yeasts, Pichia pastoris offers significant advantages over higher eukaryotic cells such as Chinese hamster ovary (CHO) or baculovirus-infected insect cell expression systems. It is easy to manipulate, has a rapid growth rate and requires inexpensive media. These greatly reduce the production time and cost, especially on a commercial scale. Unlike Saccharomyces cerevisiae, Pichia pastoris is not a strong fermentor and can be easily cultured to very high cell density of >100 g dry cell weight/liter (Siegel et al., 1989). This, plus the strong AOX1 promoter employed in driving transcription of foreign genes, have made Pichia pastoris the system of choice for high levels of expression of heterologous proteins. The AOX1 promoter also has advantages in the expression of foreign proteins that are deleterious to the expressing host because the promoter is tightly regulated and highly repressed under non-methanolic growth conditions. The inducible and tightly regulated AOX1 promoter has allowed successful expression of DT based immunotoxins, in secreted form, in Pichia pastoris strains without any mutation to confer a resistance to DT. (Woo et al., 2002). However, diphtheria toxin (DT) is a very potent toxin to all eukaryotic cells if its catalytic domain can find a route to the cytosol. Pichia pastoris is inherently sensitive to these toxins.
The prior art teaches methods for growing Pichia pastoris. For example, Pichia pastoris may be grown in a fermentor. One protocol for Pichia pastoris fermentation contains glycerol as the initial carbon source, followed by brief carbon starvation and use of methanol as the carbon source (Pichia pastoris Fermentation Using a BioFlo 110 Benchtop Fermentor, New Brunswick Scientific).
Woo et al. disclosed that, when expressing a bivalent anti-human anti-T cell immunotoxin A-dmDT390-bisFv(G4S) in Pichia pastoris, a buffered complex medium at pH 7.0 with 1% casamino acids provided the highest expression in shake flask culture and that the expression level was improved by adding PMSF in the range of 1 to 3 mM. (25 Protein Expression and Purification 270-82 (2002)).
Sreekrishna disclosed that an increased secretion level was obtained using Pichia pastoris in shake flask cultures when the cells were highly aerated and in a buffered medium at pH 6.0 that was supplemented with yeast extract and peptone (Chapter 16, Industrial Microorganisms: Basic and Applied Molecular Genetics (1993)). The growth medium contained yeast nitrogen base with ammonium sulfate, biotin and glycerol buffered to pH 6.0 with potassium phosphate buffer as well as yeast extract and peptone. The induction medium contained methanol in place of glycerol.
In contrast, the present invention provides an improved method of using Pichia pastoris to produce an immunotoxin. The immunotoxins expressed and purified in the present invention can be used in a method of inducing immune tolerance. It would be desirable to provide a method of expression and purification that increased the yield of immunotoxins. The present invention addresses this problem and others in the manner described below.